In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

[Abstract unavailable

The microtubule-binding protein tau has been implicated in the pathogenesis of Alzheimer's disease and related disorders. However, the mechanisms underlying tau-mediated neurotoxicity remain unclear. We created a genetic model of tau-related neurodegenerative disease by expressing wild-type and mutant forms of human tau in the fruit fly Drosophila melanogaster. Transgenic flies showed key features of the human disorders: adult onset, progressive neurodegeneration, early death, enhanced toxicity of mutant tau, accumulation of abnormal tau, and relative anatomic selectivity. However, neurodegeneration occurred without the neurofibrillary tangle formation that is seen in human disease and some rodent tauopathy models. This fly model may allow a genetic analysis of the cellular mechanisms underlying tau neurotoxicity.

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.

A monoclonal antibody to choline acetyltransferase (ChAT), the acetylcholine (ACh)-synthesizing enzyme, has been used to localize ChAT within neurons in immunocytochemical preparations of adult rat spinal cord. Morphological details of known cholinergic spinal neurons are presented in this study, and previously unidentified ChAT-containing neurons are also described. Immunoreaction product was present within cell bodies, dendrites, axons, and axon terminals, thereby allowing comprehensive descriptions of the distribution of ChAT-positive neurons and the interrelationships of their processes. In the ventral horn, ChAT-positive motoneurons were located in the medial, central, and lateral motor columns, and their dendrites formed elaborate longitudinal and transverse ChAT-positive bundles. These bundles were present throughout the rostrocaudal extent of the spinal cord. In the central gray matter, small ChAT-positive cell bodies were clustered around the central canal. Small longitudinal fascicles of immunoreactive processes were also observed in this region adjacent to the ependymal layer. The intermediate gray matter of virtually the entire spinal cord was spanned by medium to large ChAT-positive multipolar cells termed partition neurons. At autonomic spinal levels, partition neurons were intermingled with other immunoreactive cells that were identified as preganglionic sympathetic or parasympathetic neurons because of their locations and morphological characteristics. In the sympathetic system, four groups of ChAT-positive neurons were observed; the principal intermediolateral nucleus (ILp) in the lateral horn, the central autonomic cell column (CA) dorsal to the central canal, the intercalated nucleus (IC) located between ILp and CA, and the funicular intermediolateral neurons (ILf) in the white matter lateral to the ILp. The dendrites of ILp and CA neurons formed substantial longitudinal bundles within each group, as well as transverse bundles between the groups that resembled the rungs of a ladder. ChAT-positive cell bodies were also present in the dorsal horn, and those located in laminae III-V extended dendrites dorsally into a longitudinal plexus within lamina III.

No abstract

Although the correct architecture of epithelial tubes is crucial for the function of organs such as the lung, kidney and vascular system, little is known about the molecular mechanisms that control tube size. We show that mutations in the ATPα α and nrv2 β subunits of the Na+/K+ ATPase cause Drosophila tracheal tubes to have increased lengths and expanded diameters. ATPαand nrv2 mutations also disrupt stable formation of septate junctions, structures with some functional and molecular similarities to vertebrate tight junctions. The Nrv2 β subunit isoforms have unique tube size and junctional functions because Nrv2, but not other DrosophilaNa+/K+ ATPase β subunits, can rescue nrv2mutant phenotypes. Mutations in known septate junctions genes cause the same tracheal tube-size defects as ATPα and nrv2 mutations,indicating that septate junctions have a previously unidentified role in epithelial tube-size control. Double mutant analyses suggest that tube-size control by septate junctions is mediated by at least two discernable pathways,although the paracellular diffusion barrier function does not appear to involved because tube-size control and diffusion barrier function are genetically separable. Together, our results demonstrate that specific isoforms of the Na+/K+ ATPase play a crucial role in septate junction function and that septate junctions have multiple distinct functions that regulate paracellular transport and epithelial tube size.

No abstract